The long term goal of this ongoing study is to relate some of the defects in synaptic transmission associated with certain human presynaptic neuromuscular diseases to precise defects in nerve terminal function. 2,4- Dithiobiuret (DTB) is a thiourea derivative which causes a delayed onset neuromuscular weakness that mimics certain human neuromuscular diseases, thus providing a useful experimental model. Neuromuscular weakness induced by DTB is associated with altered release of acetylcholine (ACh) from motor nerve terminals, possible alterations of ACh receptor/ion channel complexes, and alterations of nerve terminal ultrastructure. Proposed studies continue to focus on the presynaptic mechanisms by which DTB disrupts release of ACh. Results obtained during the last funding period led to two major conclusions: a) that DTB preferentially disrupts release of ACh without affecting the release of dopamine, and 2) that DTB exerts direct effects on nicotinic receptors for ACh. In turn, this has led to the development of two testable hypotheses for the present proposal: a) that DTB disrupts quantal release of ACh by actions on the filling, mobilization, docking or fusion of cholinergic vesicles with the plasma membrane; and b) that DTB further disrupts release during high frequency or repetitive neuronal activity by actions on prejunctional nicotinic receptors at the cholinergic terminal. These hypotheses will be tested using in vitro neuromuscular preparations from rats, isolated nerve terminals (synaptosomes) of rats or cultured mouse hybridoma neuroblastoma/glioma NG108-15 cells, or rat PC12 pheochromocytoma cells. Effects of acute and chronic exposure to DTB will be examined using standard microelectrode recordings of postsynaptic potentials, ultrastructural analyses and fluorescent indicator dyes for vesicle recycling and biochemical analyses of nerve terminal and synaptic vesicle proteins. Specific questions include: 1. Does abnormal quantal release of ACh associated with DTB -induced paralysis result from changes in the normal functioning of proteins involved in quantal release of ACh from nerve terminals? 2. Does DTB alter the recycling or mobilization of cholinergic synaptic vesicles from "reserve" to "active" status? 3. Is active uptake of ACXh into the cholinergic synaptic vesicle impaired? 4. Does DTB alter the sensitivity of presynaptic nicotinic cholinergic receptors leading to impaired release of ACh? Results of these studies using this novel paralytic agent will lead to further understanding of some of the presynaptic processes of neuromuscular transmission which are perturbed in diseased states or after toxic chemical exposure, as well as increasing our understanding of how these processes interact during normal neuromuscular functioning.